A quality control system is associated with the endoplasmic reticulum (ER) which retains and selectively degrades both soluble and membrane proteins which have failed to fold, oligomerize or assemble correctly. This process assures that only fully functional proteins exit the ER and prevents delivery of a mutant protein or partially assembled receptor complex to the cell surface where it may have deleterious effects. The quality control system affects a number of proteins, including the cystic fibrosis conductance transmembrane regulator, the T cell receptor, MHC class I heavy chain, alpha-1 proteinase inhibitor, apolipoprotein B and acetyl-cholinesterase. In addition, pathogens including the human immunodeficiency virus utilize the degradative machinery to promote and conceal their infection. The overall goal of this research is to develop a detailed molecular understanding of the quality control machinery associated with ER degradation. Although the proteins destined for degradation are initially associated with the ER, the site of degradation and the machinery responsible for proteolysis remain to be identified. This proposal focuses on the degradation of an unassembled membrane spanning protein, Vph1p, in the ER of the yeast S. cervisiae. In the ER of wildtype cells, Vph1p is assembled with other subunits into an ATPase complex destined for the vacuole. However, in the absence of certain assembly proteins Vph1p is rapidly degraded in a manner that is independent of vacuolar (lysosomal equivalent) proteases and occurs in the absence of transport to the Golgi. The turnover of Vph1p exemplifies regulated degradation of membrane proteins retained in the ER and provides powerful tools with which to analyze this important phenomenon. The specific aims are: 1) to determine the precise subcellular location of Vph1p degradation using both immuno-localization and subcellular fractionation techniques; 2) to ascertain if domains of Vph1p on both sides of the ER membrane are degraded, and if so, establish whether such degradation is co-ordinated; 3) to utilize yeast strains that contain mutations in molecular chaperones, ubiquitin and/or subunits of the proteasome complex, to determine the influence of these proteins on Vph1p degradation; 4) to employ genetic approaches to isolate and characterize novel mutants that stabilize Vph1p. The identification, localization and characterization of these mutants and corresponding gene products should complement the biochemical approaches and offer insights into the process of quality control through ER associated protein degradation.